The delivery of oxygen and nutrients to the myocardium is dependent on the regulation of tone in coronary resistance vessels. Another important aspect of nutrient delivery to the myocardial tissue is blood-tissue exchange, which is regulated by many factors including permeability of exchange vessels The primary goal of this grant is to further elucidate the regulation of coronary microvascular tone and solute exchange, and to delineate underlying mechanisms of the regulatory factors. We propose to test the following hypotheses: 1) the mechanism of flow-dependent dilation involves transduction of shear stress through integrins to activate specific enzymes in endothelial cells; 2) shear stress-induced vasodilation occurs in the microcirculation of the beating heart; 3) shear stress-induced release of nitric oxide blunts the vasoconstrictor action of pressor substances in the coronary microcirculation; 4) ischemic coronary vasodilation is partially caused by carbon monoxide; 5) vasoactive substances released or carried by venules can diffuse to adjacent arterioles and induce alterations in tone; 6) cyclic GMP increases permeability through stimulation of specific kinases; 7) integrins are involved in the signaling of flow-induced increases in permeability; 8) Beta-adrenergic activation antagonizes the increase in permeability induced by NO through activation of specific phosphodiesterases. To test these hypotheses we will utilize methodologies that enable studies of the microcirculation of the beating heart and of isolated resistance and exchange vessels. Studies of the beating heart will include interventions designed to examine the preferential microvascular location(s) of shear stress-induced vasodilation under conditions of high viscosity, constant flow perfusion. The role of nitric oxide as a modulator of the pressor action of vasoconstrictors will be delineated by assessing action of the agonists before and after inhibition of nitric oxide. We will test if carbon monoxide produces ischemic coronary vasodilation by inhibiting the enzyme that induces its production. In isolated arterioles we will examine the signaling pathways involved in flow-dependent dilation by using inhibitors of tyrosine kinase, phospholipase C, and protein kinase C. Examination of pairs of isolated venules and arterioles will allow determination of shunting of vasoactive metabolites in the heart. Finally we will elucidate the mechanism of flow-induced increases in permeability of isolated venules by performing studies similar to those discussed for arterioles. We will also probe mechanisms by which cGMP influences permeability through its actions on the cytoskeleton, and the interactions between catecholamine and factors that increase permeability, by using specific inhibitors of phosphodiesterases that hydrolyze cGMP. The solutions to these hypotheses will provide unique knowledge concerning regulation of coronary microvascular regulation of coronary microvascular tone and solute exchange.